The objective of this Phase I Research Proposal is to develop affinity-based mass spectrometry assay for detection of the NIAID High Priority Biodefense Agent Staphylococcal Enterotoxin B (SEB). The assay will utilize affinity capture in pipette-fitted microcolumns modified with anti-SEB antibody to selectively retrieve SEB from buffer, tap water, and milk samples. Detection will be achieved by measuring the molecular weight of the eluted SEB via MALDI-TOF mass spectrometry. The assay is unique as it offers dual specificity: the first comes from the use of a capturing antibody, and the second comes from the observation of a specific m/z value, characteristic of the analyzed toxin, in the resulting mass spectra. The use of polyclonal antibodies in combination with MS enables capture and detection of protein toxins and related variants in a single assay, and in the process significantly reduces the cost and time of analysis. In such, the proposed assay offers significant advantages over existing and commercially available sandwich-based SEB assays. Under the three specific aims under this proposal, we will demonstrate the reproducibility and sensitivity of the SEB assay, and develop and apply quantitative approaches for detection of SEB via the proposed affinity-based mass spectrometric assay. The successful completion of these Phase I Specific Aims will result in a fully-functional SEB-MSIA assay, and will lay the foundation for an experimental/developmental Phase II program that will focus on increasing the number of pathogen assays, developing multiplexed assays capable of detecting families and cohorts of toxins, integrating data evaluation methods into the assays, and describe the use of the assays and the accompanying instrumental platform in the laboratories that are part of the CDC's Laboratory Reference Network.